PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the world?s leading infectious killer, results in 1.8 million deaths and 10.4 million new cases annually. Despite recent advancements, >40% of cases are missed with diagnostic gaps greatest in children and people living with HIV (PLWH) in whom treatment delay contributes to poor outcomes. Current TB diagnostic tests lack sensitivity in children and PLWH. Culture is often inaccessible in TB high- burden settings, has a long delay to result, and poorly predicts treatment failure and relapse. Xpert Ultra (Ultra) yields results in ~2 hours, but Ultra sensitivity is 63% in smear-negative, culture-positive adults and 67% in child TB, and relies upon difficult to collect respiratory specimens. Recognizing these limitations, we developed a stool based quantifiable PCR (qPCR) whose initial evaluations show i) a limit of detection equivalent to culture at 96 colony forming units per 50mg of stool, ii) sensitivity equivalent to sputum-based Xpert, and iii) 20- 30% increased yield amongst Xpert and culture-negative individuals with clinical TB. We are now poised to validate our findings in adults, adolescents, and children with and without HIV-infection in a multi-centered study at our sites in Swaziland, Tanzania and Mozambique. TB treatment response is monitored by symptom resolution, radiologic improvement, serial microscopy and culture. As 45% of HIV-associated and 80% of child TB is smear-negative, treatment monitoring in these key populations is limited to poorly sensitive symptomatic evaluation. Our pilot work demonstrates that persistent detection of Mtb by qPCR after 2 months of TB treatment was associated with 3-fold increased odds of treatment failure. We are now positioned to assess the treatment monitoring potential of qPCR and discriminate between participants who will fail treatment and participants who will achieve relapse free cure. TB treatment outcomes are optimized by minimizing time to appropriate treatment. Available drug susceptibility tests (Xpert and Line Probe Assay) are limited by reliance on smear-positive respiratory specimens and test placement at central laboratories. Stool-based genotypic drug susceptibility testing (DST) could provide clinicians with data to guide appropriate TB treatment, particularly in sputum smear negative patients, and avert the development of drug resistance. Building on our stool-based platform, we will assess the feasibility and test performance of LPA completed on DNA isolated from stool. Current sputum collection techniques are poorly accepted by patients, parents and health care workers. The impact of any diagnostic test is influenced by feasibility of implementation and acceptability. To comprehensively assess the potential impact of our stool-based platform, we will gather critical data regarding operational characteristics, acceptability and perceptions. Rapid, affordable tests that i) accurately diagnose TB, ii) robustly identify drug-resistance, and iii) guide treatment from accessible non-respiratory specimens could revolutionize TB care and control.